Figure 4. Bakuchiol suppresses growth of A431 xenograft tumors in nude mice.

Bakuchiol decreases viability and inhibits anchorage-independent A431 cell growth. (A, B) Bakuchiol inhibits anchorage-independent growth of A431 cells. A soft agar assay was performed with bakuchiol treatment (0, 2.5, 5, or 10 μM) and the number of colonies was counted under a microscope with the aid of the Image-Pro Plus software program (v. 6.2). Results are represented as mean values ± S.E. (n = 3). The asterisks (*, **) indicate a significant difference (p < 0.05, p < 0.01) compared with the untreated control group. (C) Bakuchiol decreases viability of A431 cells. The MTT assay was used to evaluate viability of cells treated with bakuchiol (0, 2.5, 5, or 10 μM). (D) Bakuchiol induces apoptosis of A431 cells. Apoptosis was analyzed by Annexin V/PI staining. Bakuchiol induces apoptosis in A431 cells in a dose-dependent manner. For C and D, the asterisks (**) indicate a significant difference (p < 0.01) compared with the untreated control group. (E) Bakuchiol stimulates apoptosis-related signaling pathways. A431 cells were treated with bakuchiol (0, 2.5, 5, or 10 μM) for 48 h and harvested. Immunoblotting was conducted using specific antibodies. (F) The average tumor volume of control and bakuchiol-treated mice plotted over 28 days after tumor cell inoculation. The p values indicate statistical significance for the inhibition of tumor growth by bakuchiol (**p < 0.01). (G, H) Representative images and quantification of PCNA (green) and DAPI (blue) staining of A431 xenograft tumors (n = 12). Scale bars = 100 μm. Data are represented as mean values ± S.E. The asterisks (*, **) indicate a significant (p < 0.05, p < 0.01) difference between the vehicle-treated and bakuchiol-treated groups.